In recent years, several studies have conclusively shown that numerous pathogens,
including several species in the Botryosphaeriaceae, Phomopsis, Phaeoacremonium, as well
as Phaeomoniella chlamydospora and Eutypa lata, contribute to premature decline and
dieback of grapevines. These pathogens have the ability to infect grapevines through pruning
wounds, which leads to a wide range of symptoms developing that includes stunted growth,
cankers and several types of wood necrosis. Pruning wounds stay susceptible for 2 to 16
weeks after pruning and sustained levels of pruning wound protection is therefore required.
The aims of this study were to (i) evaluate the ability of several biological agents to protect
pruning wounds, (ii) characterise unknown Trichoderma strains and identify their modes of
action and (iii) determine the optimal time of season for biological agent application.
Several biological agents were initially evaluated in a laboratory for their antagonism
against trunk disease pathogens. The best performing control agents were tested in a field
trial conducted on Merlot and Chenin blanc vines in the Stellenbosch region. Spurs were
pruned to three buds and the fresh pruning wounds were treated with benomyl as a control
treatment, Trichoderma-based commercial products, Vinevax® and Eco77®, Bacillus
subtilis, and Trichoderma isolates, USPP-T1 and -T2. Seven days after treatment the pruning
wounds were spray inoculated with spore suspensions of four Botryosphaeriaceae spp.
(Neofusicoccum australe, N. parvum, Diplodia seriata and Lasiodiplodia theobromae),
Eutypa lata, Phaeomoniella chlamydospora and Phomopsis viticola. After a period of 8
months the treatments were evaluated by isolations onto potato dextrose agar. Trichodermabased
products and isolates in most cases showed equal or better efficacy than benomyl,
especially USPP-T1 and -T2. Moreover, these isolates demonstrated a very good ability to
colonise the wound tissue.
The two uncharacterised Trichoderma isolates (USPP-T1 and USPP-T2), which were
shown to be highly antagonistic toward the grapevine trunk disease pathogens, were
identified by means of DNA comparison, and their ability to inhibit the mycelium growth of
the trunk disease pathogens by means of volatile and non-volatile metabolite production
studied. The two gene areas that were used include the internal transcribed spacers (ITS 1
and 2) and the 5.8S ribosomal RNA gene and the translation elongation factor 1 (EF). The ITS and EF sequences were aligned to published Trichoderma sequences and the percentage
similarity determined and the two Trichoderma isolates were identified as Trichoderma
atroviride. The volatile production of T. atroviride isolates was determined by placing an
inverted Petri dish with Trichoderma on top of a dish with a pathogen isolate and then sealed
with parafilm. Trichoderma isolates were grown for 2 days on PDA where after they were
inverted over PDA plates containing mycelial plugs. The inhibition ranged from 23.6% for
L. theobromae to 72.4% for P. viticola. Inhibition by non-volatile products was less than for
the volatile inhibition. Inhibition ranged from 7.5% for N. parvum to 20.6% for L.
theobromae. In the non-volatile inhibition USPP-T1 caused significantly more mycelial
inhibition than USPP-T2.
The timing of pruning wound treatment and subsequent penetration and colonisation
of the wound site was also determined. One-year-old canes of the Shiraz and Chenin blanc
cultivars were grown in a hydroponic system, pruned and spray treated with a spore
suspension of Trichoderma atroviride (USPP-T1) as well as a fluorescent pigment. On
intervals 1, 3, 5 and 7 days after treatment, the distal nodes were removed and dissected
longitudinally. From the one half, isolations were made at various distances from the pruning
surface, while the other half was observed under ultra-violet light to determine the depth of
fluorescent pigment penetration. Shortly after spray-inoculation of a fresh pruning wound,
Trichoderma was isolated only from the wound surface and shallow depths into the wound (2
to 5 mm). One week after inoculation, Trichoderma was isolated at 10 mm depths, and after 2
weeks, at 15 mm depths. Fluorescent pigment particles were observed to a mean depth of 6
mm, which suggests that initial isolation of Trichoderma at these depths was resultant of the
physical deposition of conidia deeper into the pruning wound tissue, whereas the isolation of
Trichoderma from deeper depths might be attributed to colonisation of grapevine tissue. In a
vineyard trial, fluorescent pigment was spray-applied to pruning wounds of Shiraz and
Chenin blanc grapevines during July and September at intervals 0, 1, 3, 7 and 14 days after
pruning. One week after treatment, the distal nodes were removed and dissected
longitudinally. Each half was observed under UV light and the pigment penetration
measured. For Chenin blanc and Shiraz, July pruning wounds showed significant deeper
penetration of the pigment than pruning wounds treated in September. Moreover, pruning
wounds made in September showed pigment particles in longitudinal sections up to 1 day
after pruning, whereas wounds made in July showed pigment particles up to 3 days in the
xylem vessels. These findings suggest that the best time for application of a biological
control agent should be within the first 24 hours after pruning.